Typical applications of amplicon libraries include surveying bacterial and fungal marker genes like the ribosomal 16S and 18S DNA, respectively, as well as the ITS fungal region. However, any other amplicon of interest can be examined. Modifications of this protocol are suitable for a variety of situations. For example, it can be applied to characterization of CRISPR integrations.
This method requires at least 12.5 ng of input DNA.
Oxford Nanopore sequencing can be used to sequence small genomes like bacteria, fungi, and amoeba. Nanopore sequencing can be combined with Illumina sequencing to conduct genome hybrid assembly. This approach usually results in more complete genome assemblies. With this method it is also possible to sequence relatively long stretches of RNA without going through cDNA synthesis.
The main advantage of this approach is the long reads generated but is quality is considerably lower than with Illumina sequencing. A variety of protocols are available, so please discuss with us the best approach for your research goals.
Typical applications for NexteraXT libraries include metagenomics and sequencing of small genomes like bacteria, fungi and viruses. One of the main advantages of this approach is the low input material required. It works ideally with a total of 1 ng of DNA. A final concentration of 200 pg per µl is needed; however, for accuracy during DNA quantification, we recommend providing a DNA of concentration as high as possible.
RNAseq (Total RNA)
In this method, samples from groups representing two different phenotypes are compared; ideally, a control group against an experimental one. Examples include healthy vs. diseased, wild-type vs. mutant, and two different time points from a single population of cells or tissue. The technique can be applied at the bulk level or at the single cell level. For statistical analyses purposes, three samples from each group should be provided, ideally representing biological replicates (i.e. coming from three independent experiment repeats). Technical replicates are also acceptable but less informative.
At least 100 ng of total RNA are required for bulk sequencing, and a method of purification of cells of interest is required for single cell sequencing.
This technique is analogous to RNAseq, bur specifically targets small RNAs processed by a Dicer enzyme virtue of their monophosphate groups at the 5’ end and hydroxyl groups at the 3’ termini. MicroRNAs, piwiRNAs, snoRNAs, and snRNAs can be analyzed using this method. The protocol ideally requires 1 µg of total RNA although libraries can be constructed with less material at the expense of reducing diversity in the survey.
Targeted Sequencing in Ion S5 Sequencer
This method is flexible and scalable, ideal for assessing abundance in gene panels in a cost-effective manner. The method can also be scaled up to full exomes. A variety of protocols are available, so please discuss with us the best approach for your research goals.